Medical devices, such as dermal fillers or implants made from naturally occurring materials including polysaccharides, are in direct contact with organ tissue and therefore must be made from biocompatible materials. Such materials have to (1) be inert, (2) induce little or no immune response, and (3) in some cases be degradable by the organism. Unfortunately, medical devices can be contaminated during their manufacturing, transportation and/or storage. Contamination such as by microorganisms (e.g., bacteria, virus or spores) or chemicals can induce inflammatory responses, which can lead to fever, hypotension, systemic shock and even death. Moreover, many of these materials are isolated from naturally occurring sources, such as microorganisms, and typically contain unacceptable levels of endotoxins. Therefore, the successful translation of medical devices to the clinic requires ensuring their safety until implantation.
One of the most serious contaminations that can occur in medical devices prepared from naturally occurring materials, such as polysaccharides, is endotoxins. Endotoxins are integral components of Gram-negative bacterial cell walls. Endotoxins are exogenous pyrogenic substances, i.e. they can induce fever. Endotoxins typically contain a polysaccharide chain, which is water soluble, bound to a hydrophobic or water-insoluble lipid strand. A representative structure for an endotoxin is shown in FIG. 6:
Due to their amphiphilicity, endotoxins are difficult to remove from commercially useful materials.
Several sterilization processes are commonly used to prevent contamination including filtration, gamma irradiation, ethylene oxide, steam, and chemical sterilization. These procedures are able, to varying degrees, to kill microorganisms (bacteria and mammalian cells), destroy viruses (protein carriers of genetic information such as DNA or RNA), or degrade endotoxins (lipopolysaccharides originating from bacteria wall). Autoclaving, which is a common technique used for sterilization, cannot remove endotoxins. Moreover, the removal of endotoxins from commercially useful materials, such as agarose or carrageenan, is challenging since these materials themselves are polysaccharides. Moreover, these techniques can also generate degradation products of the material, which can be toxic, or causes changes to its mechanical properties, affecting the proper operation of the device. A summary of the drawbacks of various sterilization techniques is shown in Table 1.
TABLE 1Advantages and drawbacks of the different sterilization methodsMaterial mechanicalTechniqueSterility*Endotoxinproperties preservationCostFiltration+/+/−−+$Irradiation+/+/+−−$$$Ethylene Oxide+/+/+−−$$$Steam+/+/−−−++$$Ethanol+/−/−++++$*Bacteria/Virus/Spores
Irradiation and EO are two techniques that are fast and quite efficient for removal of viruses and endotoxins. Unfortunately they cannot be used for the sterilization of polysaccharide, such as agarose, as they modify the polysaccharide backbone. Therefore specific considerations of the sterilization method have to be taken into account for obtaining a material that meets the requirements of regulatory agencies. Table 2 shows some of the requirements of the Food and Drug Administration (FDA) in the United States for clinical grade materials.
TABLE 2Some of the requirement for clinical grade materialsLevel RegulationSourceRef.Ethanol Content<0.5%v/vFDA(1)MicrobiologyN.D.FDAEndotoxins<0.05EU/mlFDA(2)(1) Serdons, et al. Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine 2008; 49: 2071(2) FDA Bacterial/Endotoxins Pyrogens. Available at http://www.fda.gov/ICECI/InspectionGuides/InspectionTechnicalGuides/ucm072918.htm#.Ug0qTDAoFos.email
Dialysis of naturally occurring materials material against pyrogen free water has been explored to overcome the limitations of the techniques discussed above. Dialysis, however, is an expensive and time consuming process.
There exists a need for improved methods for removing endotoxin from naturally occurring materials, such as polysaccharides.
Therefore, it is an object of the invention to provide for improved methods for removing endotoxin from naturally occurring materials, such as polysaccharides.
It is also an object of the invention to provide for improved methods for removing endotoxin from naturally occurring materials, such as polysaccharides, which are cost effective and can be performed over a relatively short period of time.
It is also an object of the invention to provide for improved methods for removing endotoxin from naturally occurring materials, such as polysaccharides, which are cost effective and can be performed over a relatively short period of time, and which do not significantly alter the chemical, physical, and/or mechanical properties of the material.